RESUMEN

Significant efforts have been made to improve the sialylation of recombinant glycoproteins with the aim of extending their in vivo circulation time. Here, we report a systematic functional analysis of 31 N-glycosylation-related genes on sialylation of recombinant EPO in six cell lines. BHK and CHO cells were found to sialylate recombinant EPO most effectively. None of the 31 genes, individually or in combination, was able to improve EPO sialylation in these cells. HEK293, Cos-7 and 3T3 cells showed intermediate sialylation capabilities, whereas NS0 cells sialylated recombinant EPO poorly. Overexpression of ST6GalI, ST3GalIII or ST3GalIV, but not ST3GalVI, was able to improve EPO sialylation in these four cell lines. qRT-PCR experiments revealed that ST3GalIII and ST3GalIV are indeed under expressed in HEK293, 3T3 and NS0 cells. Co-expression of upstream glycogenes failed to synergize with these sialyltransferases to further enhance sialylation, suggesting that the upstream glycogenes are all expressed at sufficient levels.

Asunto(s)

Eritropoyetina/metabolismo , Glicosilación , Silanos/metabolismo , Células 3T3/metabolismo , Animales , Células CHO , Células COS , Línea Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Células HEK293 , Humanos , Focalización Isoeléctrica , Lectinas/química , Ratones , Ácido N-Acetilneuramínico/química , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sustancias Reactivas al Ácido Tiobarbitúrico/química